The present invention is related to isolated, purified DNA sequences which can act as promoters in eukaryotic cells. More specifically, the present invention is related to such DNA sequences from tobacco which act as microspore-specific promoters and play a role in the expression of genes in microspores. This invention is also directed to chimeric genes suitable for transforming a plant comprising a structural gene under the control of the microspore-specific promoter. Further, this invention relates to plant cells and plants transformed with said chimeric gene.
In angiosperm plants, sexual reproduction requires the production of viable male and female gametophytes. Pollen, as the male gametophyte, is formed within the anther and is initiated from sporogenous cells, which develop into meiocytes. The meiocyte undergoes meiosis to form a tetrad of haploid microspores which are subsequently released into the anther locule. Following expansion and vacuolation an asymmetrical mitosis of the microspore results in bicellular pollen, containing a vegative and generative cell. In the majority of plant species, including tobacco, pollen is shed in a bicellular condition. In other plant species (e.g. cereals and Brassica sp.), pollen maturation includes the mitotic division of the generative cell such that pollen is shed in a tricellular condition.
The morphological aspects of pollen development have been studied in great detail but knowledge about the underlying molecular processes is relatively limited. Pollen formation requires coordinated gene expression in the gametophytic cells and the sporophytic tissues surrounding it. Two periods of temporal gene expression are defined in pollen development by Mascarenhas [20]. The early developmental stage starts after meiosis and ends with the microspore mitosis, the lace stage covers the period after mitosis up to mature pollen [14]. During pollen development the mRNA content present in different stages changes qualitatively [28]. Genes which are transiently and specifically transcribed during pollen development are assumed to play a crucial role in the developmental processes. Summarizing, genes involved in pollen development can be classified, according to expression pattern as early [8, 29, 35], and late genes [3, 6, 21, 32, 39, 40]. Additionally, genes transcribed in both stages have been isolated [1, 25 ,33].
This classification does not use the place of expression in consideration. If this localisation is taken as an extra criterion, the previous classification can be refined, which leads to four categories of pollen developmental genes. First, a category of genes with exclusive expression in the unicellular microspore; second, a group with localised expression in the diploid anther tissues during development; third, a group which localised expression in pollen; and fourth, a group with expression in unicellar microspores, pollen and diploid anther tissues. Genes of the first category have not been identified yet.
Previous studies of early genes restricted to microsporogenesis resulted in a group of genes of which expression [8, 29, 35] is localised in the tapetum or other anther tissues. A few genes have been characterised which are expressed in both developing pollen and in tapetum [25, 30, 33].
It is an object of the present invention to isolate and characterize a DNA sequence which is capable of acting as a microspore-specific promoter. More particularly, it is an object of the invention to isolate and characterize a microspore-specific promoter region taken from a microspore-specific gene.
The present invention relates to an isolated, purified DNA sequence, SEQ ID NO:1 from the promoter region of a microspore-specific gene of Nicotiana tabacum, upstream of a sequence encoding a protein comprising the amino acid SEQ ID NO:2 sequence set forth in FIG. 2 or a protein substantially homologous therewith.
The present invention further relates to an isolated purified DNA sequence from the promoter region of a microspore-specific gene of tobacco, the sequence consisting essentially of nucleotides xe2x88x92952 to +43 set forth in FIG. 2 or a functional fragment thereof having promoter activity.
Further this invention provides chimeric genes suitable for transforming a plant, comprising a DNA sequence as defined above and a naturally occurring or synthetic gene which is under the control of said DNA sequence.
The promoter of this invention is shown to be exclusively active in the male reproductive cell of a higher plant during early pollen development. Transcripts were observed only in the unicellular microspore. There is no expression in the sporophytic tissues that represent the majority of cells in the anther, nor in any other tissue of the plant. Expression is also developmently restricted to the unicellular microspore: the promoter is inactive during the preceding tetrad stage and the activity disappears at microspore mitosis.
Some authors reported microspore-specific genes like Bp4 [1 and PCT application WO 90/08828 ] and Bp19 [2] in Brassica napus. But the term microspore was also used for the bicellular pollen. In contrast our definition for microspore is for the unicellular stage of pollen development.
In the present Examples the BamHI-NcoI fragment consisting of nucleotides xe2x88x92952 to +43 set forth in FIG. 2 was used as promoter. The functionality of the promoter was established in a transgenic context. For this purpose the promoter was placed before the gus-reporter gene. Further it was established that the promoter is not at all active in tissues other than microspores. For this purpose the promoter was placed before the barnase gene. The ATG of the NcoI site was used as translational start. The promoter-gus and promoter-barnase cassettes were transferred in a binary plasmid and introduced in tobacco by Agrobacterium tumefaciens transformation.
As a comparison similar constructs were made with Bp4 promoter described in [1] and in WO 90/08828. The Bp4 promoter was cloned from Brassica napus using PCR amplification. Said promoter contained nucleotides 7 to 265 of FIG. 3a in WO 90/08828 and flanking HinDIII and XbaI restriction sites. Said promoter is the Bp4A promoter. FIG. 7c of WO 90/08828 discloses a 490 bp chimeric promoter of Bp4C and Bp4A. Since the Bp4A and Bp4C promoters are substantially identical, the promoter disclosed in WO 90/08828 will be functionally identical with the Bp4 promoter used in the present Examples.
The results of the comparison tests show that the present promoter is a strong and evidently microspore-specific promoter. The Bp4 promoter, however, is a second phase promoter which is only active after the first pollen mitosis. Further, the Bp4 promoter is a wean promoter as compared with the present promoter.
The present promoter importantly offers the opportunity to manipulate unicellular microspore-specific expression of genes introduced in the plant by genetic engineering. These sequences can be used to limit the expression of any given DNA-sequence to microspores and to microsporogenesis up to microspore mitosis. The microspore-specific promoter can be used to limit the expression of DNA sequences adjacent to it, to microspores in the Solanacae family and it is fully believed that these DNA sequences or functional fragments thereof will function as microspore-specific promoters in numerous other if not all microspore-bearing plant species that are capable of being genetically transformed.
These microspore-specific promoters can be used in conjunction with naturally occurring or synthetic flanking sequences critical to microspore development.
It should be noted that the identification of a promoter region is usually defined by function rather than a set DNA sequence. The expression xe2x80x9ca functional fragment thereof having promoter activityxe2x80x9d as used herein means generally a fragment of the defined promoter region sequence consisting of sufficient nucleotides upstream from the 3xe2x80x2 end of the promoter region to have promoter activity. As little as 80 to 200 bases may be sufficient DNA sequence to maintain the tissue-specificity and promoter function. It should also be recognized that some upstream DNA sequences can be arranged in opposite orientations and still retain or demonstrate enhanced promoter function. In addition, xe2x80x9cenhancer-likexe2x80x9d DNA sequences, which are usually small conserved DNA sequences ranging in size from less than 10 nucleotides to considerably larger numbers of nucleotides can also be inserted into promoter regions to enhance expression.
The chimeric gene of the invention can comprise any gene or gene fragment, whose expression product (RNA and/or protein or polypeptide) causes changes in metabolism, functioning and/or development of microspores. Such a gene may preferably be a male-sterility gene. Since the promoter Us microspore-specific, other plant functions or tissues are not affected as is shown in the Examples. Preferred male-sterility DNAs are described in EP-A-89401194.9, for example those DNAs encoding: RNases such as RNase T1 or barnase; DNases such as endonucleases (e.g., EcoRI); proteases such as papain; enzymes which catalyze the synthesis of phytohormones (e.g., isopentenyl transferase or the gene products of gene 1 and gene 2 of the T-DNA of Agrobacterium; glucanases; lipases; lipid peroxidases; plant cell wall inhibitors; or toxins (e.g., the A-fragment of diphteria toxin or botulin). Other preferred examples of male-sterility DNAs are antisense DNAs encoding RNAs complementary to genes, the products of which are essential for the normal development of fertile pollen. Further preferred examples of male-sterility DNAs encode ribozymes capable of cleaving specifically given target sequences of genes encoding products which are essential for the production of fertile pollen. Still other examples of male-sterility DNAs encode products which can render stamen cells, particularly microsporesxe2x80x94and not other parts of the plantxe2x80x94susceptible to specific diseases (e.g. fungi or virus infection) or stress conditions (e.g. herbicides).
The present invention also relates to a plant cell or plant cell cultures transformed with the chimeric gene comprising the gene the wild-type promoter of which is replaced by the promoter DNA sequence of the invention. Further, the invention relates to a plant or its seeds consisting essentially of said plant cells.